The present invention relates to a T cell factor (TCF)-responsive element, a gene and uses of the TCF-responsive element or nucleic acid construct in assays nucleic acid construct comprising a TCF-responsive element and a therapeutic and therapy.
TCFs are a family of transcription factors within the High Mobility Group (HMG) of DNA-binding proteins (Love et al., Nature, 376, 791–795,1995). The family includes TCF-1, TCF-3 and TCF-4 which are described in van der Wetering et al, (EMBO J., 10, 123–132, 1991), EP-A-0 939 122 and Korinek et al. (Science, 275, 1784–1787, 1997). TCF-4 has been shown to be involved in tumorigenesis related to Wnt/Wingless signalling. TCF and LEF-1 (lymphoid enhancer factor-1) are considered to mediate a nuclear response to Wnt signals by interacting with β-catenin. Wnt signalling and other cellular events that increase the stability of β-catenin are considered to result in transcriptional activation of genes by LEF-1 and TCF proteins in association with β-catenin. In the absence of Wnt signalling, LEF-1/TCF proteins repress transcription in association with Groucho and CBP (CREB binding protein).
In the absence of Wnt signalling, β-catenin is found in two distinct multiprotein complexes. One complex, located at the plasma membrane, couples cadherins (calcium dependent adhesion molecules) with the actin cytoskeleton whereas the other complex (containing the proteins adenomatous polyposis coli protein (APC), axin and glycogen synthase kinase 3β (GSK3β)) targets β-catenin for degradation. Wnt signalling antagonises the APC-axin-GSK3β complex, resulting in an increase in the pool of free cytoplasmic β-catenin. The free cytoplasmic β-catenin can translocate to the nucleus where it binds LEF-1/TCF factors and activates Wnt target genes. The regulation of LEF-1/TCF transcription factors by Wnt and other signals is discussed in Eastman et al, (Current Opin. Cell Biology, 11, 233–240,1999).
The APC gene is a tumour supressor gene that is inactivated in most colorectal cancers. Mutations of APC are considered to cause the accumulation of free β-catenin, which then binds TCF causing increased transcriptional activation of genes including genes important for cell proliferation (e.g. cyclin D1 (Tetsu et al., Nature 398, 422–426, 1999 and Shtutman et al., PNAS USA, 96, 5522–5527, 1999) and c-myc (He et al., Science, 281, 1509–1512,1998)). The involvement of APC in tumour development is discussed in He et al, (supra).
TCFs are known to recognise and bind TCF binding elements which have the nucleotide sequence CTTTGNN, wherein N indicates A or T (van der Wetering et al, supra).
TCF reporter genes have been constructed and are described in Korinek et al, (Science, 275, 1784–1787,1997), Morin et al, (Science, 275,1787–1790, 1997), EP-A-0 939 122 and WO 98/41631. The TCF reporter gene is said to comprise three TCF binding elements upstream of either a minimal c-Fos promoter driving luciferase expression or a minimal herpes virus thymidine kinase promoter driving chloramphenicol acetyl-transferase expression. He et al (supra) discloses TCF reporter gene constructs comprising four TCF binding elements inserted into pBV-Luc.
There is a need for an effective treatment of cancers associated with a deregulation of the Wnt signalling pathway. Such cancers include most colorectal cancers, approximately 30% of melanomas and some breast, prostate and hepatocellular carcinomas.
There is also a need for a TCF response element which when linked to an expressible gene gives improved levels of expression and specificity.